in Meat Samples Real-Time PCR Detection of Salmonella · PDF file · 2015-04-22Real-Time PCR Detection of Salmonella spp. ... MagMAX™ Express-96 Magnetic Particle Processor

USER GUIDE

ABI 29/02 – 09/10

www.afnor-validation.com

ALTERNATIVE ANALYTICAL METHODSFOR AGRIBUSINESS

For testing of Food and Environmental samples only.

Real-Time PCR Detection of Salmonella spp.in Meat SamplesUsing automated DNA isolation and magneticbead-based technologyfor use with:PrepSEQ® Nucleic Acid Extraction KitMagMAX™ Express-96 Magnetic Particle ProcessorMicroSEQ® Salmonella spp. Detection KitApplied Biosystems® 7500 Fast Real-Time PCR InstrumentRapidFinder™ Express Software

Publication Number 4485954

Revision C

The information in this guide is subject to change without notice.

DISCLAIMER

LIFE TECHNOLOGIES CORPORATION AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED ORIMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, OR NON-INFRINGEMENT. TOTHE EXTENT ALLOWED BY LAW, IN NO EVENT SHALL LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) BE LIABLE, WHETHER IN CONTRACT, TORT,WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIALDAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF.

LIMITED USE LABEL LICENSE No. 492: Environmental Testing, Quality Control/Quality Assurance Testing, Food and Agricultural Testing

Notice to Purchaser: The purchase of this product conveys to the purchaser the limited, non-transferable right to use the purchased amount of theproduct (a) to perform internal research for the sole benefit of the purchaser; and (b) for environmental testing, quality control/quality assurancetesting, food and agricultural testing, including reporting results of purchaser's activities in environmental testing, quality control/quality assurancetesting, food and agricultural testing for a fee or other commercial consideration. No other right is hereby granted expressly, by implication, or byestoppel. This product is for environmental testing, quality control/ quality assurance testing, food and agricultural testing and research purposesonly.

The purchase of this product does not grant the purchaser any additional rights, including (without limitation) the right to transfer or resell theproduct in any form or the right to use the product as a therapeutic agent or diagnostics test component. For information on obtaining additionalrights, please contact [email protected] or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California 92008.

TRADEMARKS

All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. AFNOR and NF Validation are trademarksof Association Française de Normalisation (AFNOR). Oxoid is a trademark of Oxoid Limited. Stomacher is a registered trademark of Seward Limited,UK. Whirl-Pak is a registered trademark of Aristotle Corporation. TaqMan is a registered trademark of Roche Molecular Systems, Inc., used underpermission and license.

©2014 Thermo Fisher Scientific Inc. All rights reserved.

mailto: [email protected]

Contents

About this guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Revision history . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

■ CHAPTER 1 Overview .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

■ CHAPTER 2 Enrich food samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Enrich food samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

■ CHAPTER 3 Isolate DNA with the PrepSEQ® Nucleic AcidExtraction Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Kit contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Required materials not included with the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Procedure overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Before first use of the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11Prepare Binding Solution and Wash Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Before each use of the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12Resuspend Magnetic Particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12Prepare Proteinase K Buffer Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12Prepare Lysis Buffer Premix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Set up and incubate the Lysis Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Set up the MagMAX™ Express-96 processing plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Process samples on the MagMAX™ Express-96 instrument . . . . . . . . . . . . . . . . . . . . . . . . . . 14

■ CHAPTER 4 PCR with the MicroSEQ® Salmonella spp.Detection Kit and RapidFinder™ Express Software . . . . . . . . . . . . . . . . . . . . 15

Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Kit contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Required materials not included with the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Real-Time PCR Detection of Salmonella spp. in Meat Samples User Guide 3

Important procedural guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Create or edit a run file in RapidFinder™ Express Software . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Prepare the assay beads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Set up the PCR reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Load and run the reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

View results and data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20If necessary, investigate results in SDS Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

■ CHAPTER 5 Confirmation of results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

■ APPENDIX A Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

■ APPENDIX B Supplemental information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

NF Validation™ by AFNOR certification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

PCR with Sequence Detection Software (SDS) on the 7500 Fast Real-TimePCR Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Run parameters and cycling conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27Threshold settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28Interpretation of results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Good laboratory practices for PCR and RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

■ APPENDIX C Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

■ Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Food Safety support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

Contents

4 Real-Time PCR Detection of Salmonella spp. in Meat Samples User Guide

About this guide

Revision history

Revision Date Description

C September 2014 • Clarified storage of enriched cultures and DNA samples.

• In confirmation methods, corrected incubation time for secondaryenrichment in RVS.

• Updated trademarks and copyright to Thermo Fisher Scientific.

• Other format and organizational updates.

B December 2013 • Added instructions for PCR with the MicroSEQ® Salmonella spp.Detection Kit and confirmation testing.

• Added NF Validation™ certification description.

• Changed document title to reflect additional content.

A July 2013 New document.


Overview

IMPORTANT! Before using the products described in this guide, read andunderstand the information in the "Safety" appendix in this document.

This guide describes the following NF Validation™-certified workflow for detection ofSalmonella spp. in meat samples:

1. Enrichment of 25-g meat samples in Buffered Peptone Water (BPW).2. Automated preparation of PCR-ready DNA using the PrepSEQ® Nucleic Acid

Extraction Kit and the MagMAX™ Express-96 Magnetic Particle Processor. TheMagMAX™ Express-96 Magnetic Particle Processor enables high-throughputsample processing in a 96-well format with minimal handling.

3. Real-time PCR detection of Salmonella spp. DNA in the DNA sample using theMicroSEQ® Salmonella spp. Detection Kit and RapidFinder™ Express Software onthe Applied Biosystems® 7500 Fast Real-Time PCR Instrument.

4. Confirmation testing of positive samples by an independent method.

Refer to “NF Validation™ by AFNOR certification“ on page 26 for detailedinformation about the NF Validation™ certification.

Visit www.lifetechnologies.com/foodsafety for a complete list of workflows fordetection of Salmonella spp. (Pub. no. MAN0009417).

1


http://www.lifetechnologies.com/foodsafety

Enrich food samples

Required materials

MLS: Fisher Scientific (www.fisherscientific.com) or other major laboratory supplier.

Item Source

Buffered Peptone Water (BPW) MLS

Homogenizer (Stomacher® 400 Laboratory Blender orequivalent)

Seward # 0400/001/AJ orequivalent

Homogenizer bag, as required by sample type:

With mesh, 6” × 9”, 24 oz (Whirl-Pak® Filter Bag forHomogenizer Blender, or equivalent)

Nasco # B01348WA orequivalent

No mesh, 6” × 9”, 24 oz (Whirl-Pak® Write-on Bag, orequivalent)

Nasco # B01196WA orequivalent

Enrich food samples

IMPORTANT! Use proper aseptic technique while handling samples to avoidcrosscontamination.

1. Prepare Buffered Peptone Water (BPW) according to the manufacturer’sinstructions.

2. Add 225 mL of BPW to 25 g (or 25 mL) of food sample.

2


http://www.fisherscientific.com

3. Homogenize the food sample in a homogenizer bag as described in the followingtable.Refer to the ISO 6887 standard for detailed information.A filtered bag may be used for enrichment of particulated samples.

Food type Homogenization method

Coarse food types, such as meat orpoultry[1]

Process for 1–2 minutes in ahomogenizer (Stomacher® 400Laboratory Blender with speed settingNorm, or equivalent).

Liquids or powdered foods Shake the bag at least 25 times toachieve a homogeneous suspension.

[1] Hand massage foods that cannot be processed in a homogenizer.

4. Incubate at 37±1°C under static conditions for a total incubation time of 16–20 hours.

STOPPING POINT (Optional) For convenience, enriched cultures can be stored at 5±3°Cfor up to 72 hours before proceeding to DNA isolation.

Chapter 2 Enrich food samplesEnrich food samples2


Isolate DNA with the PrepSEQ®

Nucleic Acid Extraction Kit

Product description

The PrepSEQ® Nucleic Acid Extraction Kit (Cat. nos. 4428176, 4480466) is designed forpreparation of high-quality microbial nucleic acid from broth cultures. Magneticbeads allow efficient DNA capture and sample washing. The kit enables high-throughput automation in a 96-well plate format on the MagMAX™ Express-96Magnetic Particle Processor. Visit www.lifetechnologies.com/foodsafety for a list ofworkflows with non-automated DNA isolation methods.

Kit contents and storage

Table 1 PrepSEQ® Nucleic Acid Extraction Kit

ComponentsCat. no.4480466

(100 reactions)

Cat. no.4428176

(300 reactions)Storage[1]

Magnetic Particles 2 × 1.5 mL 6 × 1.5 mL 5±3°C

Lysis Buffer 2 × 50 mL 6 × 50 mL

Roomtemperature(23±5°C)

Binding Solution (Isopropanol)[2] 1 empty bottle 3 empty bottles

Wash Buffer Concentrate[3] 2 × 26 mL 6 × 26 mL

Elution Buffer 1 × 25 mL 3 × 25 mL

Proteinase K (PK) Buffer 1 × 50 mL 3 × 50 mL

Proteinase K, 20 mg/mL 1 × 1.25 mL 3 × 1.25 mL Below –18°C

[1] Refer to the product label for expiration date.[2] Add ~35 mL of 100% isopropanol to the empty bottle before use.[3] Add 74 mL of 95% ethanol before use.

Note: Kit components may ship separately depending on configuration and storageconditions.

3



Required materials not included with the kit

Unless otherwise indicated, all materials are available from Life Technologies. MLS:Fisher Scientific (www.fisherscientific.com) or other major laboratory supplier.

Item Source

Equipment

MagMAX™ Express-96 Deep Well Magnetic ParticleProcessor

Cat. no. 4400079

96-Well Magnetic-Ring Stand Cat. no. AM10050

Block heater, 37°C MLS

Laboratory mixer, Vortex or equivalent MLS

Pipettors:

• Positive-displacement

• Air-displacement

• Multichannel

MLS

(Optional but recommended) Plate centrifuge MLS

Consumables

Disposable gloves MLS

Micropipette tips, aerosol-resistant MLS

MagMAX™ Express-96 Deep Well Plates Cat. no. 4388476

MagMAX™ Express-96 Standard Plates Cat. no. 4388475

MagMAX™ Express-96 Deep Well Tip Combs Cat. no. 4388487

(Optional) MicroAmp® Clear Adhesive Film Cat. no. 4306311

Sterile tubes, 15-mL or 50-mL MLS

Reagents

Ethanol, 95% MLS

Isopropanol, 100% MLS

Nuclease-free Water Cat. no. AM9938

Procedure overview

250 µL of enriched sample is incubated at room temperature for 10–30 minutes withProteinase K Buffer Mix. A mixture of Lysis Buffer, Binding Solution, and MagneticParticles is added, and the samples are processed on the MagMAX™ Express-96Magnetic Particle Processor through lysis, washing, and elution of the nucleic acidinto 100 µL of Elution Buffer. The eluted DNA sample is ready for downstream PCR.

Chapter 3 Isolate DNA with the PrepSEQ® Nucleic Acid Extraction KitRequired materials not included with the kit3



Set up and incubate the Lysis Plate

150 µL Proteinase K Buffer Mix (140 µL PK Buffer + 10 µL Proteinase K)+ 250 µL enriched sample

q

Incubate at room temperature 10–30 minutes

q

Set up the MagMAX™ Express-96 processing plates

Tip Comb plate: Standard plate + Deep Well Tip Comb

Elution Plate: 100 µL Elution Buffer

Wash Plate 1: 300 µL Wash Buffer

Wash Plate 2: 300 µL Wash Buffer

q

Process samples on the MagMAX™ Express-96 instrument

Select program 4428176DWPrepSEQFA, select Start, and load prepared processingplates into the instrument

q

Add 605 µL prepared Lysis Buffer Premix(250 µL Lysis Buffer + 30 µL Magnetic Particles + 325 µL Binding Solution)

to each sample and control wellin the Lysis Plate (incubation completed)

q

Load Lysis Plate, select Start, and run the program (~45 minutes)

q

DNA is in Elution Buffer (Elution Plate)

q

Proceed to PCR, or seal the plate and store the DNA below –18°C

Before first use of the kit

Before using a new PrepSEQ® Nucleic Acid Extraction Kit, prepare the reagents:• Binding Solution—Add approximately 35 mL of 100% isopropanol to an empty

Binding Solution bottle. Label the bottle to indicate that isopropanol is added.• Wash Buffer—Add 74 mL of 95% ethanol to the Wash Buffer Concentrate bottle,

and mix well. Label the bottle to indicate that ethanol is added.

Workflow

Prepare BindingSolution and WashBuffer

Chapter 3 Isolate DNA with the PrepSEQ® Nucleic Acid Extraction KitBefore first use of the kit 3


Before each use of the kit

IMPORTANT! Mix the particles vigorously before each use, to ensure that all salts aredissolved.

White precipitate occasionally forms in the Magnetic Particles tube. Extractionexperiments show that formation of precipitate does not affect performance as long asthe precipitate is fully dissolved prior to use.

1. Incubate the tube of Magnetic Particles at 37±1°C for approximately 10 minutes.

2. Vortex for approximately 10 seconds.

Note: If the white precipitate is not completely dissolved after 10 minutes at37°C, apply longer incubation times and higher temperatures (up to 50°C).

3. Keep at room temperature (23±5°C) until ready for use.

1. Combine the following components for the number of extractions required plus10% overage.

Component Volume per extraction

Proteinase K 10 µL

PK Buffer 140 µL

Total volume per extraction 150 µL

2. Mix well to disperse Proteinase K in PK Buffer.

Use Proteinase K Buffer Mix immediately or store on ice until ready to use.

1. Combine the following components for the number of extractions required plus10% overage.

Component Volume per extraction

Lysis Buffer 250 µL

Magnetic Particles[1] 30 µL

Binding Solution (isopropanol) 325 µL

Total volume per extraction 605 µL

[1] Resuspended and thoroughly mixed.

2. Mix well and store at room temperature (23±5°C).

Store Lysis Buffer Premix at room temperature for up to 2 hours before use. Mix wellbefore dispensing.

ResuspendMagnetic Particles

PrepareProteinase KBuffer Mix

Prepare LysisBuffer Premix

Chapter 3 Isolate DNA with the PrepSEQ® Nucleic Acid Extraction KitBefore each use of the kit3


Set up and incubate the Lysis Plate

1. Collect the enriched sample from the incubator and briefly mix the samples.

2. Thoroughly mix the prepared Proteinase K Buffer Mix if it has been stored on ice.

3. Set up the Lysis Plate in a MagMAX™ Express-96 Deep-Well Plate according tothe following table.Pipet up and down 2–3 times to mix after addition of enriched sample orNuclease-free Water to the Proteinase K Buffer Mix.

Component Sample well NEC well[1]

Prepared Proteinase K Buffer Mix 150 µL 150 µL

Enriched sample 250 µL —

Nuclease-free Water — 250 µL

[1] Reserve at least one well per plate containing Nuclease-free Water as a negative extraction control.

4. Incubate the Lysis Plate at room temperature (23±5°C) for 10–30 minutes.

Note: During this incubation, set up the MagMAX™ Express-96 processingplates as described in the following section.

Set up the MagMAX™ Express-96 processing plates

Set up the MagMAX™ Express-96 processing plates as described in the followingtable.

Plate MagMAX™ Express-96plate type Action

Tip Comb Standard Place a 96-well Deep Well Tip Comb ina standard plate.

Elution Plate Standard Add 100 µL of Elution Buffer to eachsample and control well.

Wash Plate 1 Deep Well Add 300 μL of Wash Buffer to eachsample and control well.

Wash Plate 2 Deep Well Add 300 μL of Wash Buffer to eachsample and control well.

Chapter 3 Isolate DNA with the PrepSEQ® Nucleic Acid Extraction KitSet up and incubate the Lysis Plate 3


Process samples on the MagMAX™ Express-96 instrument

1. Power on the MagMAX™ Express-96 Magnetic Particle Processor, select program4428176DWPrepSEQFA using the Up-arrow and Down-arrow keys, and pressStart.

2. Load the prepared plates according to the readout on the instrument, verifyingthat their orientation is {A1 to A1}.

Plate Action

Tip Comb Load the tip comb, then press Start.

Elution Plate Load the Elution Plate, then press Start.

Wash Plate 1 Load Wash Plate 1, then press Start.

Wash Plate 2 Load Wash Plate 2, then press Start.

3. When incubation of the Lysis Plate is complete:a. Vortex the prepared Lysis Buffer Premix for approximately 5 seconds to

ensure even distribution of the Magnetic Particles.

b. Add 605 µL of Lysis Buffer Premix to each sample and control well of theLysis Plate.

4. Load the Lysis Plate into the instrument and press Start.

5. When DNA sample preparation is complete (~45 minutes; “Enjoy your DNA” isdisplayed on the screen), remove the Elution Plate from the instrument. The Elution Plate contains DNA in Elution Buffer.

6. Proceed directly to real-time PCR, or store the DNA in one of the following ways:• 5±3°C for up to 24 hours.• Below –18°C for long-term storage.

Note: If the Elution Plate contains Magnetic Particles, place the Elution Plate ona 96-Well Magnetic Ring Stand for ≥1 minute before collecting the eluate for PCR.

Chapter 3 Isolate DNA with the PrepSEQ® Nucleic Acid Extraction KitProcess samples on the MagMAX™ Express-96 instrument3


PCR with the MicroSEQ® Salmonellaspp. Detection Kit and RapidFinder™

Express Software

Product description

The MicroSEQ® Salmonella spp. Detection Kit detects Salmonella spp. simply, reliably,and rapidly in food samples using a lyophilized reagent format. The assay uses thepolymerase chain reaction (PCR) to amplify a unique microorganism-specific DNAtarget sequence and a TaqMan® probe to detect the amplified sequence.

The MicroSEQ® assay beads contain all the components necessary for the real-timePCR reaction: Salmonella spp.-specific probe and primers, enzyme, and other buffercomponents. The assay beads also contain an internal positive control (IPC) probe,primers, and template, to monitor for PCR inhibition. Pathogen Detection NegativeControl is included in the kit. Unknown samples and positive control samples areprovided by the investigator.

RapidFinder™ Express Software provides step-by-step instructions to set up the real-time PCR assays followed by automated data analysis. Online help is provided withinthe software. The software is designed for use on the Applied Biosystems® 7500 FastReal-Time PCR Instrument.

Kit contents and storage

Table 2 MicroSEQ® Salmonella spp. Detection Kit [96 reactions; Cat. nos. 4403930, 4412639 (includes thePrepSEQ® Nucleic Acid Extraction Kit)]

Component Description Amount Cap color Storage

MicroSEQ® Salmonellaspp. Detection Kit

Salmonella spp. Assay Beads,8-tube strips

12 strips (96 tubes)

1 rack

Green (rack) 5±3°C

Protect fromlight andmoisture.[1]MicroAmp® Optical 8-Cap

Strips12 strips (96 caps) N/A

Pathogen DetectionNegative Control

Pathogen Detection NegativeControl

1.5 mL Red 5±3°C

[1] Excessive exposure to light may affect the fluorescent probes. To protect the beads from moisture, do not remove the desiccant from the pouch, and seal the pouch tightly each time you remove assay bead strips.

Note: Kit parts may ship separately, depending on the configuration ordered andstorage conditions.

4


Required materials not included with the kit

Unless otherwise indicated, all materials are available from Life Technologies. MLS:Fisher Scientific (www.fisherscientific.com) or other major laboratory supplier.

Item Source

Instrument and equipment

Applied Biosystems® 7500 Fast Real-Time PCRInstrument

Contact your local LifeTechnologies representative.

7500 Fast Precision Plate Holder for MicroAmp®

Tube StripsCat. no. 4403809

MicroAmp® 96-Well Base Cat. no. N8010531

MicroAmp® Cap Installing Tool Cat. no. 4330015

MicroAmp® Multi-removal Tool Cat. no. 4313950

Benchtop microcentrifuge with 8-tube strip adapter

or

Plate centrifuge

MLS

Laboratory mixer (Vortex mixer or equivalent) MLS

Pipettors:

• Positive-displacement

• Air-displacement

• Multichannel

MLS

Consumables

Aerosol-resistant pipette tips MLS

Disposable gloves MLS

MicroAmp® Fast 8-Tube Strip, 0.1-mL Cat. no. 4358293

MicroAmp® Optical 8-Cap Strip, 300 strips Cat. no. 4323032

Reagents

Nuclease-free Water Cat. no. AM9938

Chapter 4 PCR with the MicroSEQ® Salmonella spp. Detection Kit andRapidFinder™ Express SoftwareRequired materials not included with the kit

4



Workflow

Create or edit a run file in RapidFinder™ Express Software

q

Prepare the assay beads

q

Set up the PCR reactions

q

Load and run the reactions

q

View results and data analysis

Important procedural guidelines

IMPORTANT! Seal the tubes with the transparent, optical strip-caps provided in thekit. Always use intact 8-cap strips, even if empty tubes have been added next toreaction tubes.

Do not use colored caps or tubes for real-time PCR reactions. Colored caps or tubesmay affect dye-signal readings during real-time PCR.

• RapidFinder™ Express Software determines the plate layout and therefore mustbe set up before distributing DNA samples for the real-time PCR.

• Follow these instructions to ensure proper storage of the tube strips:– Cut the storage pouch at the notch above the resealable strip.– Always reseal the storage pouch with desiccant, and replace at 5±3°C.

• If DNA samples were stored before PCR, thaw (if necessary), vortex, andcentrifuge at 1000–2000 × g for approximately 1 minute, to remove anycondensation from the adhesive film before opening the plate (to avoid crosscontamination).

Chapter 4 PCR with the MicroSEQ® Salmonella spp. Detection Kit andRapidFinder™ Express Software

Workflow

4


• Follow these additional tips when setting up the PCR:– 8-tube strips can be cut apart with scissors.

If necessary, trim any remaining connector material from the cut to allow abetter fit against adjacent tubes in the 7500 Fast Precision Plate Holder forMicroAmp® Tube Strips.

– MicroAmp® Tube Strips are labeled 1–8 on the side of the tubes, to orienttube strips during handling.

Figure 1 MicroAmp® Tube Strip labelingIn this photo, the embossed numbers have been enhanced for visual clarity.

If necessary for visual reference from above, mark the tab at one end of thecap strip. Do not mark any of the caps (this could interfere with real-timePCR detection).

– Use a new pipette tip for each sample.– If you mix the assay beads with the DNA samples by pipetting up and

down, keep the pipette tip at the bottom of the tube to minimize aerosolformation and cross-contamination.

– Use the MicroAmp® 96-Well Base and the MicroAmp® Cap Installing Tool toseal the assay tubes with the optical cap strips. This will avoid collapsing,bending, or misaligning the tubes.Confirm that the strips are straight and that each tube is in line with theadjacent tube.

– Use a plate adapter for vortexing the tube strips, or hold the strips in theMicroAmp® 96-Well Base while vortexing.

– Follow the recommendations in “Good laboratory practices for PCR and RT-PCR“ on page 28.

Create or edit a run file in RapidFinder™ Express Software

On the main page of the RapidFinder™ Express Software, select Create/Edit a RunFile , and select the target pathogen, number of samples, replicates, and positiveand negative controls for each target at the prompts.The software determines the sample layout based on the information entered, andcreates a run file.

Chapter 4 PCR with the MicroSEQ® Salmonella spp. Detection Kit andRapidFinder™ Express SoftwareCreate or edit a run file in RapidFinder™ Express Software

4



Follow the plate layout determined by the RapidFinder™ Express Software.

1. Transfer the appropriate number of individual tubes or 8-tube strips from thestorage pouch to a 96-well base at room temperature (23±5°C).

2. If required by the plate layout, place empty MicroAmp® Fast 8-Tube Strips (orpartial strips) to balance the tray when the assay tubes are placed in theinstrument later.

Set up the PCR reactions

For step-by-step instructions, in RapidFinder™ Express Software v1.1, select PipetteSamples on the main page.

1. If necessary, thaw samples and controls completely, and mix each sample orcontrol thoroughly.If the DNA samples have been stored, see “Important procedural guidelines“ onpage 17.

2. Following the layout determined by RapidFinder™ Express Software, add 30 µLof sample or control to each assay bead at room temperature (23±5°C), and mixby gently pipetting up and down a few times.Beads dissolve in 1–5 seconds.Alternatively, vortex the assay tubes after they are capped in the final step.

3. Seal the tubes with the transparent, optical cap strips provided in the kit.

4. Make sure that the reactions are thoroughly mixed: if reactions were notpreviously mixed during the pipetting step, vortex to mix.

5. Make sure that the reagents are at the bottom of tubes: briefly centrifuge the tubestrips at 200–600 × g for about 20 seconds.



4


Load and run the reactions

In the RapidFinder™ Express Software, select Start Instrument Run on the mainpage, select the appropriate run file, and follow the software prompts.

1. Transfer the tubes to the instrument in the same configuration as the run layout.Use the 7500 Fast Precision Plate Holder for MicroAmp® Tube Strips in theinstrument.

2. Close the tray to the instrument, and follow the RapidFinder™ Express Softwareprompts to start the run.

Figure 2 Transfer tubes to the 7500 Fast instrumentRapidFinder™ Express Software directs the user to load empty strip tubes in column 1 (far left)and column 12 (far right), if needed. The empty capped 8-tube strips evenly distribute theclamping load applied to the sample tube strips during processing, thereby minimizing the riskof collapsing any tubes.


In the RapidFinder™ Express Software, select View Results on the main page,select the appropriate run file, and follow the prompts to view results.Data analysis is automated by the software.

Chapter 4 PCR with the MicroSEQ® Salmonella spp. Detection Kit andRapidFinder™ Express SoftwareLoad and run the reactions

4


Follow the RapidFinder™ Express Software prompts for "Investigating WarningResults or Failed Runs in the SDS Software."

IMPORTANT! If you modify a RapidFinder™ Express Software run file in the SDSSoftware, you cannot open the run file again in the RapidFinder™ Express Software.To avoid altering a RapidFinder™ Express Software run file, save the run file under anew name in the SDS software before performing any actions.

1. From View Results in the RapidFinder™ Express Software, select and open therun file, and then click View in SDS.

2. Select File4Save As, and save the run file under a new name.

If necessary,investigate resultsin SDS Software



4


Confirmation of results

In the context of NF Validation™ certification, all samples identified as positive by theMicroSEQ® Salmonella spp. Detection Kit must be confirmed by one of the followingtests:

• Perform a second enrichment step in RVS (0.1 mL BPW in 10 mL RVS broth):incubate at 41.5±1°C for 6–27 hours, then streak onto XLD agar and anotherselective agar, and incubate at 37±1°C for 24±3 hours. Perform a Latex test(Oxoid™ FT0203A ) on the observed characteristic colonies.

Note: In the case of a negative Latex test, perform a biochemical gallery onpurified colonies of Salmonella. If the confirmatory test remains negative, performa second selective enrichment in MKTTn broth, and follow the confirmatory testsdescribed in the CEN or ISO standardized methods.

• Use any other method certified NF Validation™ whose principle must be differentfrom that of the MicroSEQ® Salmonella spp. Detection Kit. The detection protocolof the second validated method used for the confirmation shall be followedentirely. Steps that are previous to the confirmation shall be common to bothmethods (that is, enrichment in BPW at 37°C).

In the event of discordant results (presumptive positive with the alternative method,not confirmed by one of the means described above), the laboratory must follow thenecessary steps to guarantee the validity of the obtained result.

5


Troubleshooting

Observation Possible cause Recommended action

Eluate volume from DNAsample preparation is less than90 µL

Evaporation during the elutionstep.

If three 30-µL PCR reactions are required, addElution Buffer to the eluate to bring the volumeto ~100 µL before proceeding to PCR.

Remove the Elution Plate from the MagMAX™

Express-96 as soon as processing is complete,to minimize evaporation.

Inhibition of downstream PCR,indicated by nondetection ofIPC reaction

Magnetic Particles were in theElution Plate.

Avoid disturbing the Magnetic Particles duringtransfer of eluted DNA to the lyophilized assay.

Avoid transfer of Magnetic Particles using oneof the following methods (optional):

• Place the Elution Plate on the 96-WellMagnetic Ring Stand during transfer ofeluted DNA sample to the lyophilizedassay.

• Spin the plate at maximum speed in aplate centrifuge for the equivalent ofapproximately 4,000 × g for approximately30 seconds, to pellet the MagneticParticles to the bottom of the plate.

Elution Plate containsincompletely removedparticulate residue from thefood sample.

Avoid residue during transfer of eluted DNA tothe lyophilized assay.

(Optional) Spin the plate at maximum speed ina plate centrifuge for the equivalent ofapproximately 4,000 × g for approximately30 seconds, to pellet the food residue to thebottom of the plate.

In positive control wells, no IPCsignal is detected, but target-specific signal is detected.

A high copy number of targetDNA exists in samples,resulting in preferentialamplification of the target-specific DNA.

No action is required. The result is consideredpositive.

In positive control wells, notarget-specific signal isdetected.

Positive control was omitted(pipetting error).

Repeat the assay. Make sure to pipet thepositive control into all positive control wells.

A



In negative control wells, noIPC signal is detected, but atarget-specific signal isdetected.

Carryover contaminationcaused target signal in negativecontrol wells.

Additionally, no IPC signal innegative control wells can becaused by:

• A high copy number oftarget DNA exists insamples, resulting inpreferential amplificationof the target-specific DNA.

• A problem occurred withIPC amplification.

To correct carryover contamination, repeat theassay using fresh aliquots of all reagents andclean pipetting equipment.

To determine whether IPC amplification is aproblem, examine unknown wells for an IPCsignal. If an IPC signal is present, IPCamplification is not a problem.

In negative control wells,target-specific signal isdetected.

Carryover contaminationoccurred.

1. Repeat the assay using fresh aliquots ofall reagents and clean pipettingequipment.

2. If the negative control continues to showcontamination, repeat the assay using anew kit.

3. If the negative control continues to showcontamination, contact TechnicalSupport.

In unknown wells, no IPC ortarget-specific signal isdetected.

Inhibition of PCR occurred. Dilute the sample 1:5 with Nuclease-freeWater to dilute PCR inhibitors, and repeat theassay. If PCR remains inhibited, repeat thesample preparation.

Refer to other troubleshooting suggestions forremoval of Magnetic Particles or particulateresidue from the DNA sample.

In unknown wells, no IPC signalis detected, but target-specificsignal is detected.

A high copy number of targetDNA exists in samples,resulting in preferentialamplification of the target-specific DNA.

No action is required. The result is consideredpositive.

Multicomponent plot signalsfor FAM™, VIC®, and ROX™

detectors increase/decreasesduring cycles 1–15, but theamplification curve and resultare not affected (thisobservation applies to View inSDS mode).

Incomplete mixing anddissolution of the lyophilizedbead with sample or control.

After addition of 30 µL of sample or PathogenNegative Control to the bead and capping thetubes:

1. Vortex strips at high speed for about10 seconds, and centrifuge the strips at200–600 × g for about 10 seconds.

2. Vortex the strips again on high speed forabout 10 seconds, and centrifuge thestrips at 200–600 × g for about 1 minute.

Ensure that all liquid is at the bottom of thetubes and the beads are fully dissolved beforeproceeding.

Appendix A TroubleshootingA



Replicate results for a sampleare inconsistent.

All replicate wells for a sampledo not have the same result.

If more than two replicates yield the sameresult (for example, 2 of 3 replicates arenegative, but 1 replicate is positive), refer toyour laboratory protocol to determine whetherto repeat the assay using fresh samples andreagents.

If only 2 replicates were run and the resultsare not consistent, repeat the assay usingfresh samples and reagents.

Amplicon contamination. • Contamination wasintroduced into the PCRclean area from post-amplification reactiontubes that were eitheropened in the clean areaor brought into the PCRclean area fromcontaminated gloves orsolutions.

• Contamination wasintroduced into the real-time PCR instrument fromcrushed and broken PCRreaction tubes.

To confirm amplicon contamination, performthe following experiment:

Prepare negative control samples using atleast one 8-tube strip of MicroSEQ® AssayBeads.

1. Divide the assay beads into two sets.

a. To the first set of assay beads, add30 μL of Nuclease-free Water.

b. To the second set of assay beads,add 29 μL of Nuclease-free Waterplus 1 μL of 1 U/μL Uracil DNAGlycosylase (Cat. no. 18054-015).

2. Run samples on the 7500 Fast Real-TimePCR Instrument using SDS software andselect Fast 7500 run mode.

3. Under the instrument tab:

• Select Add Step to stage 1 of the PCRcycle that consists of 10 minutes at50°C.

• Extend the 95°C step from20 seconds to 10 minutes.

Amplicon contamination is indicated by target-specific signal in the –UNG samples and notarget-specific signal in +UNG samples.

If the instrument block was contaminated,consult the 7300/7500/7500 Fast Real-TimePCR System Absolute Quantitation UsingStandard Curve Getting Started Guide (Pub.no. 4347825) and/or contact a servicerepresentative to clean the instrument.

Appendix A Troubleshooting A


Supplemental information

Specificity

The MicroSEQ® Salmonella spp. Detection Kit can detect all Salmonella enterica speciestested and did not detect any non-Salmonella species tested. The genus Salmonellaconsists of the two species Salmonella enterica and Salmonella bongori. Salmonella entericaincorporates the most important clinical serovars for humans. The method does notallow detection of Salmonella bongori.

NF Validation™ by AFNOR certification

Visit www.lifetechnologies.com/foodsafety for a complete list of workflows fordetection of Salmonella spp. (Pub. no. MAN0009417).

Table 3 NF Validation certification of the workflow

Certification Expiration

ABI 29/02 – 09/10

www.afnor-validation.com

ALTERNATIVE ANALYTICAL METHODSFOR AGRIBUSINESS

For information about theexpiration date of theNF Validation™ certification,refer to the certificate, ABI29/02 – 09/10, available at www.afnor-validation.com or www.lifetechnologies.com/foodsafety.

The MicroSEQ® Salmonella spp. Detection Kit has been certified “NF Validation™”.The certification uses the ISO 16140 standard for the validation of alternative methods(Alternative Analytical Methods for Agribusiness. Certified NF Validation™; www.afnor-validation.com). This kit was compared and found equivalent to theISO 6579 reference method. The validated workflow described in this user guideincludes:

• Enrichment in BPW• The PrepSEQ® Nucleic Acid Extraction Kit• The MicroSEQ® Salmonella spp. Detection Kit• The Applied Biosystems® 7500 Fast Real-Time PCR Instrument

B



http://www.afnor-validation.com



http://www.afnor-validation.com

• RapidFinder™ Express Software• Confirmation testing as described in Chapter 5, “Confirmation of results“

Table 4 Validated matrices

Reference method Matrix

NF EN ISO 6579 (2002): Horizontal methodfor the detection of Salmonella spp.

Meat products (processed andunprocessed): Poultry, pork, and beef

General remarks and recommendations:• Comply with Good Laboratory Practices (GLP; refer to EN ISO 7218 standard).• Follow ISO 6579 and ISO 6887 standards for the preparation of initial

suspensions.• In the context of NF Validation™ certification, samples of more than 25 grams

have not been tested.

PCR with Sequence Detection Software (SDS) on the 7500 Fast Real-Time PCR Instrument

This section includes run parameters, cycling conditions, and threshold values forSDS Software v1.4 on the 7500 Fast Real-Time PCR Instrument. For detailedinstructions on setup and programming the instrument, refer to the guideaccompanying your instrument or to the 7300/7500/7500 Fast Real-Time PCR SystemAbsolute Quantitation Using Standard Curve Getting Started Guide (Pub. no. 4347825).

Table 5 Run parameters for the 7500 Fast instrument

Assay Absolute Quantitation (Standard Curve)

Container 96-Well Clear

Template Blank Document

Run Mode 7500 Fast

Sample Volume 30 µL

Table 6 Assay probe reporter dyes and quenchers

Target Reporter Quencher

Salmonella spp. FAM™ none

IPC VIC® none

Run parametersand cyclingconditions

Appendix B Supplemental informationPCR with Sequence Detection Software (SDS) on the 7500 Fast Real-Time PCR

InstrumentB


Table 7 Thermal cycler settings for the 7500 Fast instrument

StageStage 1

(Enzyme activation)Stage 2(PCR)

Rep. 1 (Hold)40

Denature Anneal/extend

Temp. 95°C 95°C 60°C

Time 2 min 3 sec 30 sec

Samples with a FAM™ CT value <35.69 are considered positive, when using thefollowing recommended instrument threshold settings:

Auto or Manual CT Manual

FAM™ (Salmonella spp.) detector threshold 0.5

VIC® (IPC) detector threshold 0.3

Baseline Auto

The following table is a basic guide for interpretation of results:

FAM™ dye signal(Salmonella spp.) VIC® dye signal (IPC) Interpretation

+ +, – Positive

– + Negative

– – See Appendix A,“Troubleshooting“

Good laboratory practices for PCR and RT-PCR

When preparing samples for PCR or RT-PCR amplification:• Wear clean gloves and a clean lab coat (not previously worn while handling

amplified products or used during sample preparation).• Change gloves whenever you suspect that they are contaminated.• Maintain separate areas and dedicated equipment and supplies for:

– Sample preparation and reaction setup.– Amplification and analysis of products.

• Do not bring amplified products into the reaction setup area.• Open and close all sample tubes carefully. Avoid splashing or spraying samples.• Keep reactions and components capped as much as possible.

Thresholdsettings

Interpretation ofresults

Appendix B Supplemental informationGood laboratory practices for PCR and RT-PCRB


• Use a positive-displacement pipettor or aerosol-resistant barrier pipette tips.• Clean lab benches and equipment periodically with 10% bleach solution or

DNAZap™ Solutions (Cat. no. AM9890).

For additional information, refer to ISO 22174 (2005).

Appendix B Supplemental informationGood laboratory practices for PCR and RT-PCR B


Safety

WARNING! GENERAL SAFETY. Using this product in a manner not specifiedin the user documentation may result in personal injury or damage to theinstrument or device. Ensure that anyone using this product has receivedinstructions in general safety practices for laboratories and the safetyinformation provided in this document.

· Before using an instrument or device, read and understand the safetyinformation provided in the user documentation provided by themanufacturer of the instrument or device.

· Before handling chemicals, read and understand all applicable Safety DataSheets (SDSs) and use appropriate personal protective equipment (gloves,gowns, eye protection, etc). To obtain SDSs, see the “Documentation andSupport” section in this document.

C


Chemical safety

WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards,ensure laboratory personnel read and practice the general safety guidelines forchemical usage, storage, and waste provided below, and consult the relevantSDS for specific precautions and instructions:· Read and understand the Safety Data Sheets (SDSs) provided by the

chemical manufacturer before you store, handle, or work with any chemicalsor hazardous materials. To obtain SDSs, see the “Documentation andSupport” section in this document.

· Minimize contact with chemicals. Wear appropriate personal protectiveequipment when handling chemicals (for example, safety glasses, gloves, orprotective clothing).

· Minimize the inhalation of chemicals. Do not leave chemical containers open.Use only with adequate ventilation (for example, fume hood).

· Check regularly for chemical leaks or spills. If a leak or spill occurs, followthe manufacturer's cleanup procedures as recommended in the SDS.

· Handle chemical wastes in a fume hood.· Ensure use of primary and secondary waste containers. (A primary waste

container holds the immediate waste. A secondary container contains spillsor leaks from the primary container. Both containers must be compatiblewith the waste material and meet federal, state, and local requirements forcontainer storage.)

· After emptying a waste container, seal it with the cap provided.· Characterize (by analysis if necessary) the waste generated by the particular

applications, reagents, and substrates used in your laboratory.· Ensure that the waste is stored, transferred, transported, and disposed of

according to all local, state/provincial, and/or national regulations.· IMPORTANT! Radioactive or biohazardous materials may require special

handling, and disposal limitations may apply.

Biological hazard safety

WARNING! BIOHAZARD. Biological samples such as tissues, body fluids,infectious agents, and blood of humans and other animals have the potential totransmit infectious diseases. All work should be conducted in properlyequipped facilities using the appropriate safety equipment (for example,physical containment devices). Safety equipment also may include items forpersonal protection, such as gloves, coats, gowns, shoe covers, boots,respirators, face shields, safety glasses, or goggles. Individuals should betrained according to applicable regulatory and company/ institutionrequirements before working with potentially biohazardous materials. Followall applicable local, state/provincial, and/or national regulations. The followingreferences provide general guidelines when handling biological samples inlaboratory environment.

· U.S. Department of Health and Human Services, Biosafety in Microbiologicaland Biomedical Laboratories (BMBL), 5th Edition, HHS Publication No. (CDC)21-1112, Revised December 2009; found at:

Appendix C SafetyChemical safety C


www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf· World Health Organization, Laboratory Biosafety Manual, 3rd Edition,

WHO/CDS/CSR/LYO/2004.11; found at:www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf

Appendix C SafetyBiological hazard safetyC


http://www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf

http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf

Documentation and support

Customer and technical support

Visit www.lifetechnologies.com/support for the latest in services and support,including:

• Worldwide contact telephone numbers• Product support, including:

– Product FAQs– Software, patches, and updates

• Order and web support• Product documentation, including:

– User guides, manuals, and protocols– Certificates of Analysis– Safety Data Sheets (SDSs; also known as MSDSs)

Note: For SDSs for reagents and chemicals from other manufacturers,contact the manufacturer.

Food Safety support

Website: www.lifetechnologies.com/foodsafety

Support email: [email protected]

Phone number in North America: 1-800-500-6855

Phone number outside of North America: Visit www.lifetechnologies.com/support,select the link for phone support, and select the appropriate country from thedropdown menu.

Limited product warranty

Life Technologies Corporation and/or its affiliate(s) warrant their products as set forthin the Life Technologies' General Terms and Conditions of Sale found on LifeTechnologies' website at www.lifetechnologies.com/termsandconditions. If you haveany questions, please contact Life Technologies at www.lifetechnologies.com/support.


http://www.lifetechnologies.com/support


mailto:[email protected]


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References

ISO. 2002. Microbiology of food and animal feeding stuffs – Horizontal method forthe detection of Salmonella spp. Reference number 6579:2002.

ISO. 1999. Microbiology of food and animal feeding stuffs – Preparation of testsamples, initial suspension and decimal dilutions for microbiological examination –Part 1: General rules for the preparation of the initial suspension and decimaldilutions. Reference number 6887-1.

ISO. 2003. Microbiology of food and animal feeding stuffs -- Preparation of testsamples, initial suspension and decimal dilutions for microbiological examination --Part 2: Specific rules for the preparation of meat and meat products. Referencenumber 6887-2.


For support visit lifetechnologies.com/support or email [email protected]

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Documents

in Meat Samples Real-Time PCR Detection of Salmonella · PDF file · 2015-04-22Real-Time PCR Detection of Salmonella spp. ... MagMAX™ Express-96 Magnetic Particle Processor